Epidepride, (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxybenzamide+ ++, the iodine analogue of isoremoxipride (FLB 457), was found to be a very potent dopamine D2 receptor antagonist. Optimal in vitro binding required incubation at 25 degrees C for 4 h at pH 7.4 in a buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. Scatchard analysis of in vitro binding to striatal, medial frontal cortical, hippocampal and cerebellar membranes revealed a KD of 24 pM in all regions, with Bmax's of 36.7, 1.04, 0.85, and 0.37 pmol/g tissue, respectively. The Hill coefficients ranged from 0.91-1.00 in all four regions. The IC50's for inhibition of [125I]epidepride binding to striatal, medial frontal cortical, and hippocampal membranes for SCH 23390, SKF 83566, serotonin, ketanserin, mianserin, naloxone, QNB, prasozin, clonidine, alprenolol, and norepinephrine ranged from 1 microM to greater than 10 microM. Partial displacement of [125I]epidepride by nanomolar concentrations of clonidine was noted in the frontal cortex and hippocampus, but not in the striatum. Scatchard analysis of epidepride binding to alpha 2 noradrenergic receptors in the frontal cortex and hippocampus revealed an apparent KD of 9 nM. At an epidepride concentration equal to the KD for the D2 receptor, i.e. 25 pM, no striatal alpha 2 binding was seen and only 7% of the specific epidepride binding in the cortex or hippocampus was due to binding at the alpha 2 site. Correlation of inhibition of [3H]spiperone and [125I]epidepride binding to striatal membranes by a variety of D2 ligands revealed a correlation coefficient of 0.99, indicating that epidepride labels a D2 site. In vitro autoradiography revealed high densities of receptor binding in layers V and VI of prefrontal and cingulate cortices as well as in striatum. In vivo rat brain uptake revealed a hippocampal:cerebellar and frontal cortical:cerebellar ratio of 2.2:1 which fell to 1.1:1 following haloperidol pretreatment. These properties suggest that [125I]epidepride is a superior radioligand for the in vitro and in vivo study of striatal and extrastriatal dopamine D2 receptors.